Somatic cloning is the process of creating genetically identical plants from plant tissue other than male and female gametes. In one approach to somatic cloning, plant tissue is cultured in an initiation medium that includes hormones, such as auxins and/or cytokinins, to initiate formation of embryogenic tissue, such as embryonal suspensor masses, that are capable of developing into somatic embryos. The embryogenic tissue is then further cultured in a multiplication medium that promotes establishment and multiplication of the embryogenic tissue to form pre-cotyledonary embryos (i.e., embryos that do not possess cotyledons). The pre-cotyledonary embryos are then cultured in a development medium that promotes development and maturation of cotyledonary somatic embryos which can, for example, be placed on germination medium to produce germinants, and subsequently transferred to soil for further growth, or alternatively, placed within manufactured seeds and sown in soil where they germinate to yield seedlings.
The multiplication (maintenance) stage of somatic cloning of plant tissue in the laboratory is typically carried out in liquid suspension cultures in shake flasks using a batch method, also known as splitting. In the practice of a batch culture method, embryogenic tissue is cultured in liquid multiplication medium for a period of time; the embryogenic tissue is separated from the multiplication medium (e.g., by allowing the embryogenic tissue to settle out of the medium); then aliquots of the embryogenic tissue are removed and introduced into separate volumes of fresh multiplication medium for further culture. This process is repeated as often as desired to yield a multiplicity of containers that each include separate batches of the embryogenic tissue culture. In addition to small volumes and multitudes of containers, it is difficult to control the growth conditions in shake flasks, and there is culture variability between flasks.
Although the batch culture method is useful at laboratory scale, it is impractical to use the batch method for commercial-scale production of somatic embryos. Bioreactors are more suitable for large-scale production and provide several advantages over the shake-flask, batch method, including automation, and the ability to more closely monitor and control the culture environment, such as pH, sugar concentration, dissolved oxygen, and carbon dioxide, resulting in more homogeneous cultures and higher yield of quality somatic embryos than the shake-flask method.
The successful operation of commercial-scale liquid bioreactors for the multiplication of plant embryogenic tissue requires automatic regulation of the biomass concentration. In a production environment, success is achieved by quickly multiplying vigorous embryogenic tissue. A production friendly approach for multiplying plant embryogenic tissue is in a fed-batch bioreactor, where a small volume of plant embryogenic tissue and multiplication media is inoculated into the bioreactor and additional multiplication media is added over time until a sufficient volume of plant embryogenic tissue (biomass) has been achieved or the maximum volume of the bioreactor is reached.
Regulation of the biomass concentration is important as there are strong correlations between biomass concentration and the concentration of media components and extra cellular products in the bioreactor. There are two broad classes of extra-cellular components, those that induce somatic embryogenesis and those that inhibit it. It is well known that a minimum biomass concentration is needed to induce somatic embryogenesis, while it has also been shown that at high biomass concentrations, somatic embryogenesis is inhibited. Key media components that are inversely proportional to biomass concentration are sugar and plant hormones. At high biomass concentrations, both classes of media components can become depleted. The sugar concentration is important as it is the primary osmotic agent in liquid multiplication media.
In order to regulate biomass concentration, multiplication media should be added at a rate that best matches the growth of the biomass. However, this is difficult as the actual growth rate of the biomass may deviate significantly from a priori estimates, and under exponentially increasing biomass growth conditions, the biomass concentration can run away. Another difficulty is that a fed-batch reactor can be inoculated with a small amount of culture and fed media over a long period of time, e.g., multiple weeks, which can lead to significantly under or over feeding the biomass in the bioreactor.
The present invention provides methods of multiplying plant embryogenic tissue in a bioreactor.